PB0390 - Identification of key regulators of procoagulant COAT platelet generation by quantitative phosphoproteomic analysis and phosphoflow

Background: Upon combined activation by Collagen-And-Thrombin (COAT), a fraction of platelets lose their aggregating properties and become procoagulant. Decreased or enhanced procoagulant platelet generation lead to bleeding or thrombotic events, respectively. The intracellular signalling underlying this dichotomous activation is only partially described.

Aims: We investigated whether key regulators of the procoagulant response can be identified with time-lapse phosphoproteomic analysis and subsequently validated by a flow cytometry based intracellular staining technique (phosphoflow).

Methods: Platelets were activated at RT with convulxin and thrombin in presence or absence of calcium, which generated procoagulant or aggregating phenotypes, respectively. Platelets were sampled at baseline and different time points up to 8 min after activation. The phosphoproteomes were analysed by Tandem-Mass-Tag and Mass Spectrometry. Protein-specific phosphorylation sites of interest were monitored at different time points in both aggregant and procoagulant phenotypes by phosphoflow cytometry.

Results: We identified over 7200 different phosphorylation sites (phosphosites) corresponding to 1886 unique proteins, out of which 1643 (87%) showed significant regulation upon stimulation. Our data indicate that during the procoagulant response proteins are gradually dephosphorylated (and hyper-phosphorylated during aggregation) (Fig.1). We identified 65 antithetically regulated phosphosites in the two activation end-points at 8 min. Among these, we observed an antithetical phosphorylation status of sodium-calcium-exchanger (NCX) at Serine382 and Serine381 in procoagulant versus aggregating platelets. This observation is in line with our previous data showing a critical functional role of NCX for the generation procoagulant platelets (Thromb Haemost;2021;121:309). A differential phosphorylation status was also observed at Serine239 of the vasodilator stimulated phosphoprotein (VASP). This downregulation in procoagulant platelets was measured by phosphoflow, which confirmed the results from the phosphoproteomic analysis (Fig.2).

Conclusion(s): The present study highlights the utility of phosphoproteome analysis to detect time-dependent changes of key molecular regulators of the dichotomous response leading to the generation of procoagulant platelets besides aggregating ones.